Rice Genetics IV - IRRI books - International Rice Research Institute

Progress of sequencing in the RGPWe are currently equipped with 14 units of ABI3700 capillary type-sequencers usedfor massive routine sequencing and 10 ABI377 96-well slab-gel-type sequencers usedmainly for gap filling and PAC-end sequencing. Robotic machines are employed forplasmid isolation, sequencing reaction, colony picking, or clone transfer from onetiter plate to another. For the first assembly of 4,000 shotgun sequence data, eachchromatogram file is assembled with computer software, Phred/Phrap or its equivalent,Tracetuner. The assembled data are then visualized by another computer software,Consed. If sequence gaps are detected after assembly or if the score of eachnucleotide is too low to satisfy a reliable threshold value even after assembly, sequencesin these regions must be reanalyzed. In case of gaps, first, a shotgun clonebridging the gap must be located using the Phred/Phrap data, and then this cloneshould be sequenced after preparing subclones by a shotgun or deletion method. Anotherchoice is to use primer walking by designing PCR primers for a Dye-terminatorsequencing reaction using the bridging clone as a template. The former method ischeaper and saves more time than the latter.The RGP is now focusing on chromosome 1 to complete its sequence. The size ofthis chromosome is estimated at 52 Mb based on its total genetic distance. PCR screeningof the 34,560 PAC clones with about 400 STS/EST markers and a computationalsearch of the flanking sequences of the sequenced PAC/BAC among 120,000 BAC-STCs resulted in 52 contigs covering 27 Mb of chromosome 1 as of August 2000. Asmentioned above, the strategy adopted in the RGP to make a sequence-ready physicalmap largely depends on STS/EST markers; therefore, the genomic region withoutsuch markers must be filled by a combination of STC search and fingerprints. Thedisadvantage of the latter method is the length of time required to obtain sequenceinformation on PAC/BAC to be used for the STC search (about three weeks from thebeginning of PAC/BAC culturing to the end of the first shotgun sequencing). However,this is the only method that can be used for filling gaps between genomic regionswithout any markers and much effort must be given to this approach.By the end of September 2000, 48 PACs on chromosome 1, 12 PACs on chromosome6, 2 PACs on chromosome 2, 1 PAC on chromosome 3, and 1 PAC on chromosome8 had been completely sequenced and annotated. Following these clones, 25PACs and 2 BACs on chromosome 1, 7 PACs on chromosome 6, and 1 PAC on chromosome2 had also been completely sequenced and are under annotation. The totalsequenced length is 13.0 Mb including the overlapped regions within a contig. Thesesequences have been registered in the DNA Databank of Japan (DDBJ) and the informationis freely available on the Web.We observed several characteristics of the completed and annotated sequences.The average predicted gene density is one gene in every 5 kb. This ratio suggests thepresence of about 80,000 genes within the rice genome, assuming their even distribution.The total number of expressed rice genes was previously estimated at about20,000–30,000 based on the matching frequency of genomic and expressed genes sofar cloned. This large discrepancy in the estimated total gene number could be due toThe International Rice Genome Sequencing Project: . . . 193